1. Field of the Invention
The invention relates to recombination systems and methods for eliminating nucleic acid sequences from the genome of eukaryotic organisms, and to transgenic organisms—preferably plants—comprising these systems.
2. Description of the Background
The purpose of biotechnological research into organisms consists in, inter alia, obtaining commercially utilizable information on the function of certain genes and gene products and the elucidation of biosynthetic pathways or disease mechanisms. The information obtained in this manner can be employed in a multiplicity of ways. They serve for example for the production of novel medicaments, the development of alternative, biotechnological production methods or the generation of modified plants. An aim of biotechnological research into plants is the generation of plants with advantageous novel characteristics, for example for increasing agricultural productivity, improving the quality in foodstuffs or for the production of certain chemicals or pharmaceuticals (Dunwell J M, J Exp Bot. 2000; 51 Spec No: 487-96).
In the generation of transgenic organisms, selection of the organisms which have been modified in the desired manner is required owing to the poor efficacy of the methods used (such as, for example, stable transformation or, in particular, homologous recombination). Transgenic plants can be generated by a series of techniques (Review: Potrykus I. and Spangenberg G. ed. (1995) Gene transfer to plants. Springer, Berlin). In particular the gene transfer mediated by Agrobacterium tumefaciens and the bombardment of plant cells with the particle gun play an important role in this context. An important problem is the fact that transgenic DNA, once stably introduced into an organism, can only be removed with difficulty. The genes for resistance to antibiotics or herbicides, which are used during the transformation procedure for selection purposes, remain in the transgenic plants, which contributes substantially to the lack of acceptance of these “gene food” products among consumers.
It has therefore been attempted for some time to develop techniques by means of which foreign DNA can be integrated into the plant genome at the specific sites or reexcised therefrom (Ow D W and Medberry S L (1995) Crit Rev in Plant Sci 14:239-261).
The skilled worker is familiar with a variety of systems for the site-directed removal of recombinantly introduced nucleic acid sequences. They are based on the use of sequence—specific recombinases and two recognition sequences of said recombinases which flank the sequence to be removed. The effect of the recombinase on this construct brings about the excision of the flanked sequence, one of the recognition sequences remaining in the genome of the organism. Various sequence-specific recombination systems are described, such as the Cre/lox system of the bacteriophage P1 (Dale E C and Ow D W (1991) Proc Natl Acad Sci USA 88:10558-10562; Russell S H et al. (1992) Mol Gene Genet 234: 49-59; Osborne B I et al. (1995) Plant J. 7, 687-701), the yeast FLP/FRT system (Kilby N J et al. (1995) Plant J 8:637-652; Lyznik L A et al. (1996) Nucleic Acids Res 24:3784-3789), the Mu phage Gin recombinase, the E. coli Pin recombinase or the R/RS system of the plasmid pSR1 (Onouchi H et al. (1995) Mol. Gen. Genet. 247:653-660; Sugita Ket al. (2000) Plant J. 22:461-469). Here, the recombinase (for example Cre or FLP) interacts specifically with its corresponding recombination sequences (34 bp lox sequence and 47 bp FRT sequence, respectively) in order to delete or invert the interposed sequences. Reports on successful applications of these systems in plants are limited. Thus, David Ow's group has demonstrated that a selection marker used for the transformation of plants which was flanked by two lox sequences can be reexcised from the plant genome by the expression of Cre (Dale E C and Ow D W (1991) Proc Natl Acad Sci USA 88:10558-10562). A disadvantage of the sequence-specific recombination systems is the reversibility of the reaction, that is to say an equilibrium exists between excision and integration of the marker gene in question. This frequently brings about the selection of mutations, i.e. as soon as a mutation blocks the further interaction of the lox recognition sequences with the enzyme, the (undesired) product is removed from the equilibrium and fixed. This not only applies to the Cre-lox system, but also to the other sequence-specific recombinases (see above). A further disadvantage is the fact that one of the recognition sequences of the recombinase remains in the genome, which is thus modified. This may have effects on the characteristics of the organisms when, for example, the recognition sequence modifies or destroys reading frames or genetic control elements such as promotors or enhancers. Furthermore, the recognition sequence which remains in the genome excludes a further use of the recombination system, for example for a second genetic modification, since interactions with the subsequently introduced recognition sequences cannot be ruled out. Substantial chromosomal rearrangements or deletions may result.
Zubko et al. describe a system for the deletion of nucleic acid sequences from the tobacco genome, where the sequence to be deleted is flanked by two 352 bp attP recognition sequences from the bacteriophage Lambda. Deletion of the flanked region takes place independently of the expression of helper proteins in two of eleven transgenic tobacco lines by spontaneous intrachromosomal recombination between the attP recognition regions. The disadvantages of this method are that recombination, or deletion, cannot be induced specifically at a particular point in time, but takes place spontaneously. The fact that the method worked only in a small number of lines suggests that the integration locus in the cases in question tends to be unstable (Puchta H (2000) Trends in Plant Sci 5:273-274).
On page 12 in the key to FIG. 32, WO 96/14408 describes a method for eliminating a genetic locus in which in each case one recognition sequence of the homing restriction endonuclease I-SceI is inserted at the respective end of the sequence to be deleted. Treatment with the endonuclease leads to double-strand breaks at both ends of the sequence to be deleted. The free ends then join up by means of “recombination”. The “recombination” cited here can only be an illegitimate recombination—as can also be seen from the Figure—(for example a non-homologous end-joining (NHEJ) event), since no homologous sequences exist at the two remaining ends of the genomic DNA. Illegitimate recombination, however, leads to unpredictable recombination events. This may have effects on the characteristics of the organisms if for example reading frames or genetic control elements such as promotors or enhancers are modified or destroyed thereby. The system requires two recognition sequences which flank the fragment to be deleted.
The generation of sequence-specific double-strand breaks with the aid of restriction enzymes in eukaryotic genomes such as yeast (Haber J E (1995) Bioassays 17:609-620), mammalian cells (Jasin M (1996) Trends Genet. 12:224-228) or plants (Puchta H (1 999a) Methods Mol Biol 113:447-451) is described.
What is described is the induction of an intramolecular recombination on a plasmid DNA in Xenopus oocytes by sequence-specific cleavage with the endonuclease I-SceI (Segal D J and Caroll D (1995) Proc Natl Acad Sci USA 92:806-810) or by synthetic, chimeric nucleases (Bibikova M et al. (2001) Mol Cell Biol 21(1):289-297). The aim is the site-directed recombination between two homologous sequences between which a suitable nuclease cleavage site is located. Both cases are extrachromosomal recombination events in which in each case only part of the extra chromosomal plasmid DNA undergoes homologous recombination.
Posfai et al. describe a method for exchanging genes in the prokaryote E.coli (Posfai G et al. (1999) Nucleic Acids Res 27(22):4409-4415). Here, recombination between the endogenous and the mutated gene results in the E.coli genome, induced by cleavage with the restriction enzyme I-SceI. Aim and object was the exchange of an endogenous gene for a mutated transgene. Recombinations in E.coli proceed in a markedly simpler way and with greater efficacy than in higher eukaryotes (for example described by Kuzminov A (1999) Microbiol Mol Biol Rev. 63(4):751-813).
Dürrenberger et al. describe the induction of recombination in chloroplasts of the single-celled green alga Chlamydomonas reinhardtii using the I-SceI homing endonuclease (Dürrenberger F et al. (1996) Nucleic Acid Res 24(17):3323-3331). Recombination takes place between the endogenous 23S gene and an inserted 23S cDNA which contains a I-SceI cleavage site. Double-strand breaks are induced by mating the transgenic organism in question with an organism expressing I-SceI. Recombinations in chloroplasts proceed in a markedly simpler manner and with greater efficacy than in the chromosomal DNA of higher eukaryotes. Thus, indeed, homologous recombination appears to be the preferred, normal way of DNA integration in plastids (chloroplasts) (described in: Heifetz P B and Tuttle A M (2001) Curr Opinion Plant Biol 4:157-161). It appears that plastids have a specific system which enables them to undergo homologous recombination, as opposed to the nucleus, and facilitates the site-directed introduction of foreign DNA (Heifetz P B (2000) Biochimie 82:655-666).
The gene targeting technique, in which a site-directed integration into the chromosomal DNA of the host organism is to be achieved by means of homologous recombination works acceptably well only in the case of prokaryotes and yeast. The generation of corresponding transgenic organisms is possible in a few species only (such as, for example, mice) and even then highly complicated (see also Kanaar R Hoeijmakers J H (1997) Genes Funct 1(3):165-174). The existing, poor homologous recombination efficacy (approx. 1:1×106) is compensated for in this case by the use of complicated, sophisticated selection techniques which are limited to the species in question (such as, for example, “ES” cell technology). In other species—but above all in Higher Plants—such technologies have not been established as yet (Mengiste T and Paszkowski J (1999) Biol Chem. 380:749-758; Vergunst A C and Hooykaas P J J (1999) Crit Rev Plant Sci 18:1-31; Puchta H (1999) Methods Mol Biol 113:447-451; Hohn B and Puchta H (1999) Proc Natl Acad Sci USA 96:8321-8323). Attempts to achieve homologous recombination in plants resulted in random, nonhomologous “illegitimate” insertion events in most cases. Here, the DNA introduced is integrated at one or more unpredictable sites in the plant genome. Integration takes place by means of illegitimate recombination (Roth D B and Wilson J H (1988) illegitimate recombination in mammalian cells. In “Genetic recombination”, R. Kucherlapati and G. R. Smith Edts., American Society of Microbiology, Washington, USA; pp.621-635) and not in sequence regions which are homologous to the transferred DNA (Puchta H and Hohn B (1996) Trends Plant Sci. 1:340-348). The problem of lacking efficacy in homologous recombination, which is serious predominantly in plants, is generally known to the skilled worker. The causes are addressed by current research (Review article: Mengiste T and Paszkowski J (1999) Biological Chemistry 380(7-8):749-58). Increasing the efficacy of homologous recombination has long been a need in plant biotechnology which is hitherto unresolved.
A further need which has long existed in biotechnological research and which is not addressed by any of the established systems is the provision of systems and methods which enables the site-directed elimination of nucleic acid sequences from the chromosomal DNA of a eukaryotic organism and allow the repeated application to the same organism. For example, it is an aim of plant biotechnology further to improve by means of recombinant methods existing high-yielding varieties. In this context, it is particularly important to eliminate, after the transformation has taken place, superfluous transgene sequences such as selection markers. In addition, methods for the predictable elimination of sequences, for example from the chromosomal DNA of an organism, would offer further applications in the field of genetic engineering which are of great interest scientifically and economically.